Polyhydroxyalkanoate levels as an indicator of bioreactor health

ABSTRACT

A method has been developed to monitor the health of an activated sludge environment in a wastewater process comprising monitoring the levels of polyhydroxyalkanoates (PHA) produced and correlating those levels with various selected sample parameters. In general, levels of PHA in excess of about 15% to about 20% dry weight of the biomass is an indication that the biocatalytic efficiency of the wastewater treatment process is impaired.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/231,025 filed Sep. 8, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for monitoring andcontrolling biological activity in wastewater and controlling thetreatment thereof. Specifically a method has been developed thatcorrelates the production of polyhydroxyalkanoates (PHA) with bioreactorhealth and biocatalytic efficiency.

BACKGROUND OF THE INVENTION

[0003] A number of devices and systems to process and purify water fromindustrial operations and municipal sources prior to discharging thewater are known. Activated-sludge wastewater treatment plants, which arewell known in the art, have been most often utilized to address thisproblem. Additionally, many industrial and municipal water treatmentplants utilize biological systems to pre-treat their wastes prior todischarging into the usual municipal treatment plant. In theseprocesses, the microorganisms used in the activated sludge break down ordegrade contaminants for the desired water treatment. Efficient processperformance and control requires quick and accurate assessment ofinformation on the activity of microorganisms. This has proven to be adifficult task in view of the wide variety of materials and contaminantsthat typically enter into treatment systems. Variations in the quantityof wastewater being treated, such as daily, weekly or seasonal changes,can dramatically change numerous important factors in the treatmentprocess, such as pH, temperature, nutrients and the like, the alterationof which can be highly detrimental to proper wastewater treatment.Improperly treated wastewater poses serious human health dangers. It isimperative therefore to maintain the health and biocatalytic efficiencyof these activated sludge systems.

[0004] Various biological processes are currently used in wastewatertreatment plants to assist in contamination degradation. In a typicalprocess, contaminants in the wastewater, such as carbon sources(measured as biological oxygen demand or BOD), ammonia, nitrates,phosphates and the like are digested by the activated sludge inanaerobic, anoxic and aerobic stages, also known in the art. In theanaerobic stage, the wastewater, with or without passing through apreliminary settlement process, is mixed with return activated sludge.

[0005] The goal of wastewater bioreactors is to mineralize inlet organicand inorganic compounds (nitrogen oxides e.g., nitrate, nitrite, andammonia) to carbon dioxide and nitrogen gas resulting in a cleaneffluent stream. The efficiency of industrial wastewater treatmentsystems is especially important since loss of performance/capacity totreat process wastewater translates to lower manufacturing up time.Presently, there are no rapid methods to assess the biocatalyticcapacity of wastewater reactors.

[0006] Currently, crude macroscopic parameters are used to gauge theperformance of wastewater bioreactors. These crude macroscopicparameters include: exit carbon as measured by COD (chemical oxygendemand) or TOC (Total Organic Carbon); exit nitrogen by Total KjeldahlNitrogen (TKN) and exit phosphate by Ion Chromotography. Whilemeasurements of effluent leakage of exogenously supplied carbon, such asmethanol and organic acid, could be used to identify an impairedbioreactor, these compounds would not be reliable indicators ofbioreactor health because many other processes can impact the amount ofcarbon removed. Furthermore, assessment of performance using thesemetrics results in a responsive operating strategy, i.e., changes toreactor loads that are made only after deviation from the desiredperformance is observed. Finally, the catalytic activity of the biomasspresent in the bioreactors is time consuming to measure. For example,the denitrification rate is determined by removing biomass from thereactor and performing a batch rate study; taking about 1-2 days toperform, and therefore cannot be used to gauge the currentdenitrification capacity of the reactor. These batch studies are usefulin establishing long term performance characteristics. To date, therehave been no reports describing the relationship between cell physiologyand catalytic capacity wastewater bioreactors.

[0007] The above methods are useful for monitoring the health ofactivated sludge systems however they contain several drawbacksincluding the inability to accurately predict a reduction or loss ofdenitrification activity in the system before nitrate or one of thedenitrification intermediates is present in the bioreactor. This resultsin nitrate leakage or incomplete denitrification that is highlydetrimental and undesirable to such systems. An improved method oftracking biocatalytic efficiency is needed, particularly with respect todenitrification potential.

[0008] The problem to be solved, therefore is to provide a facile,highly responsive method of monitoring activated sludge environments torapidly predict loss of denitrification activity and other indicators ofbiocatalytic efficiency such as the concentrations of nitrate, ammonia,sulfate, phosphate and carbon dioxide in the system.

SUMMARY OF THE INVENTION

[0009] The present invention provides reliable methods to monitorbioreactor health and to maintain viable cultures within the bioreactor.Specifically, Applicants have solved the above-stated problem by makingthe correlation between the production of an internal storage moleculeand denitrification rate as a control strategy. This internal storagemolecule may be a class of storage molecules, collectively termedpolyhydroxyalkanoates (PHA), or glycogen or the like. Preferably, thisinternal storage molecule is polyhydroxyalkanoates (PHA). The PHA levelin the bacteria of the activated sludge can be easily measured andcontrolled by the level of nutrients (ammonia, phosphate, sulfate) inthe bioreactor.

[0010] Specifically, the present invention provides a method formonitoring and controlling the biocatalytic efficiency of a wastewatertreatment process comprising: a) providing an activated sludgeenvironment comprising:

[0011] (i) a carbon influx;

[0012] (ii) cultures of autotrophic, heterotrophic and facultativemicroorganisms;

[0013] (iii) feed nutrients; and

[0014] (iv) an end electron acceptor;

[0015] b) sampling wastewater from anaerobic, anoxic and/or aerobicstages of the treatment process; c) measuring the concentration ofpolyhydroxyalkanoates present in the sample to determine the status ofselected sample characteristics; and d) adjusting the feed nutrients inthe activated sludge environment depending on the status of the selectedsample as measured in c), whereby the biocatalytic efficiency of awastewater treatment process is controlled.

[0016] An indication that the biocatalytic efficiency of the wastewatertreatment process is impaired is seen when the polyhydroxyalkanoatesconcentration is greater than about 15 to about 20 dry weight percent ofthe biomass. Preferably, the feed nutrients are adjusted accordingly inthe activated sludge environment when the PHA concentration is fromabout 10 to about 20 dry weight percent of the biomass. More preferably,the feed nutrients are adjusted accordingly in the activated sludgeenvironment when the PHA concentration is from about 10 to about 15 dryweight percent of the biomass.

[0017] Sample characteristics are selected from the group consisting ofefficiency of denitrification, nitrate concentration, ammoniaconcentration, sulfate concentration, phosphate concentration and carbondioxide concentration. Similarly, feed nutrients are selected from thegroup consisting of nitrate, ammonia, sulfate, sulfide, urea andphosphate. In addition, an end electron acceptor is selected from thegroup consisting of oxygen, nitrate, nitrite, nitrous oxide, ferricoxide, and sulfate.

[0018] The invention additionally provides a method of maintainingviable cultures in an activated sludge environment in the absence ofcarbon influx comprising: a) providing an activated sludge environmentcomprising:

[0019] (i) a carbon influx;

[0020] (ii) cultures of autotrophic, heterotrophic and facultativemicroorganisms;

[0021] (iii) feed nutrients; and

[0022] (iv) an end electron acceptor;

[0023] b) removing the feed nutrients from the activated sludgeenvironment while continuously monitoring the concentration ofpolyhydroxyalkanoates present in the activated sludge environment; c)removing the carbon influx from the activated sludge environment whenthe concentration of polyhydroxyalkanoates is greater than about 15 toabout 20 dry weight percent of the biomass; and d) adding a minimalconcentration of nitrate to the activated sludge environment of step c);whereby the cultures of autotrophic, heterotrophic and facultativemicroorganisms are maintained in a viable state in the absence of acarbon influx.

[0024] In a preferred embodiment, the carbon influx is removed from theactivated sludge environment when the concentration of PHA is about 20dry weight percent of biomass or greater.

[0025] In another preferred embodiment, the minimal concentration ofnitrate added to the activated sludge environment of step c) is based onthe COD content of the PHA in the biomass. Specifically, the CODprovided by the PHA is equal to the fraction of PHA times the total drybiomass times fraction of carbon in PHA times the conversion factor forTOC to COD and is represented by Equation 1.

PHA COD(mg/L)=(0.2 mg PHA/mg MLSS)×[MLSS(mg/L)]×(0.556 mg C/mgPHA)  EQUATION 1:

[0026] The amount of nitrate that can be added to the activated sludgeenvironment is equal to the COD provided by the PHA multiplied by theamount of nitrate-N reduced/COD)×(4.5 mg nitrate/mg nitrate-N).

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 shows a Macroscopic Reactor Performance without Ammonia.Nitrate load in mg/d (closed circle), Nitrate exiting reactor in mg/L(squares). Lines are drawn to indicate data trends. Arrow 1: Switch frommethanol rich feed to organic acids rich feed. Arrow 2: First reactorshut down. Arrow 3: Second reactor shut down.

[0028]FIG. 2 shows the Reactor Performance over Time in days withoutAmmonia. 2A: NUR vs. time with organic acid rich feed composition ascarbon source. (Arrow 1: Switch from methanol rich feed to organic acidsrich feed. Arrow 2: First reactor shut down. Arrow 3: Second reactorshut down.) 2B: NUR vs. time with methanol as carbon source. 2C: Nocarbon control. 2D: PHA concentration in percent biomass dry weight.NUR=mg nitrate-N/mg MLVSS-min.

[0029]FIG. 3 shows the correlation between Nitrate Uptake Rate (NUR) andPHA Level. NUR with organic acid rich feed composition as carbon source(closed circles). NUR with methanol as carbon source (X). The hatchedline represents a linear regression of the organic acid rich feedcomposition data.

[0030]FIG. 4 shows the Macroscopic Reactor Performance with AmmoniaAddition. Nitrate load in mg/d (closed circle), Nitrate exiting reactorin mg/L (open square). Lines are drawn to illustrate data trends.

[0031]FIG. 5 shows the Reactor Performance over Time with AmmoniaAddition. 5A: NUR vs. time with organic acid rich feed composition ascarbon source. 5B: NUR vs. time with methanol as carbon source. 5C: Nocarbon control. 5D: PHA concentration in percent biomass dry weight.NUR=mg nitrate-N/mg MLVSS-min.

[0032]FIG. 6 shows the internal PHA as a carbon source. Closed circlesrepresent PHA weight fraction of the cell mass. Closed squares representthe nitrate concentration in the reactor. Lines are drawn to indicatethe trends.

[0033]FIG. 7 shows the PHA accumulation during ammonia limitation.Squares represent the nitrate load to the reactor, circles represent theresidual nitrate in the reactor and diamonds represent the weightpercent of PHA in the biomass.

[0034] The invention can be more fully understood from the followingdetailed description.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention is useful for monitoring and controllingbioreactor health and bioreactor catalytic efficiency in an activatedsludge environment by correlating the level of denitrification in thesludge with the production of an internal storage molecule. Thisinternal storage molecule may be polyhydroxyalkanoates (PHA), glycogen,or the like. Preferably, this internal storage molecule ispolyhydroxyalkanoates (PHA).

[0036] The method of the invention comprises providing an activatedsludge environment that comprises a number of elements including acarbon influx, a variety of autotrophic, heterotrophic and facultativemicroorganisms, feed nutrients and at least one type of end electronacceptor to support respiration by the heterotrophic bacteria.Wastewater from the activated sludge system is then sampled from any ofthe anaerobic, anoxic, and/or aerobic stages of the bioreactor and theconcentration of the internal storage molecule(s) is measured in thesample's bacteria according to methods well known in the art. Apreferred method of measuring PHA levels is the method described by Riisand Mai, 1988 (Journal of Chromatography, 445:285-289) as outlined infrain the General Methods section of the Examples. Glycogen levels can bedetermined according to the methods described within Gerhardt et al.(Manual of Methods for General Bacteriology, American Society forMicrobiology, Washington, D.C., 1981). Levels of PHA in excess of15%-20% dry weight percent of the biomass indicate that the biocatalyticefficiency of the wastewater treatment process is impaired, and thatdenitrification potential in particular may be compromised. Feednutrients may be adjusted accordingly to reverse the imbalance.Preferably, the feed nutrients are adjusted accordingly in the activatedsludge environment when the PHA concentration is from about 10 to about20 dry weight percent of the biomass. More preferably, the feednutrients are adjusted accordingly in the activated sludge environmentwhen the PHA concentration is from about 10 to about 15 dry weightpercent of the biomass.

[0037] In this disclosure, a number of terms and abbreviations are used.The following definitions are provided.

[0038] The term “biocatalytic efficiency” will mean the specific removalrate of the target compound e.g., grams of organic removed per grams ofbiomass per time. Biocatalytic efficiency is generally consideredimpaired when the levels of PHA within the biomass are in excess ofabout 15 to about 20 dry weight percent of the biomass.

[0039] The term “denitrification” relates to a biological anaerobicprocess that occurs under reduced conditions in which nitrate acts asthe electron acceptor to convert nitrate or nitrite to gaseous products.Many bacteria are able to perform this denitrification process to firstreduce nitrate to nitrite, and then nitrite to nitric oxide (NO),nitrous oxide (N₂0), and finally nitrogen gas (N₂). Within a wastewateractivated sludge environment, denitrification or nitrate reduction is akey process that is required to effectively treat wastewater prior toeffluent release.

[0040] The term “denitrification efficiency” will mean the extent ofreduction of nitrate or nitrite to the desired end product dinitrogen(N₂).

[0041] The term “denitrification potential” will mean the rate ofreduction of nitrate to dinitrogen by a unit of biomass.

[0042] The term “wastewater treatment process” will mean a biologicalreactor system comprising an activated sludge environment that has theability to degrade various organic compounds and other persistentpollutants. As used herein the term “activated sludge environment” willmean the mixture of wastewater, bacteria, and nutrients needed tomaintain the health of the bacteria and effect the biocatalyticdegradation of organic molecules.

[0043] The term “carbon influx” will mean the composition of organicmaterial entering into a wastewater treatment process. The carbon influxwill typically contain a variety of aromatic and straight chain organicmolecules from industrial processing streams. With regard to thecomposition of organic material entering into a wastewater treatmentprocess as carbon influx, this influx may comprise organic compoundsselected from the group consisting of amines, alcohols, organic acids,carbohydrates, proteins, and amino acids.

[0044] The term “selected sample characteristics” refers to thecharacteristics of a wastewater sample that is tested for PHAconcentrations. Selected sample characteristics typically define thehealth of a wastewater system and will relate to concentrations ofvarious nutrients, including but not limited to nitrate, ammonia,sulfate, sulfide, urea, phosphate and carbon dioxide.

[0045] The term “feed nutrients” means various nutrients necessary tomaintain the health and biocatalytic efficiency of the activated sludgeenvironment. The components that comprise a wastewater system generallyinclude an organic source (e.g., carbohydrates, organic acids andalcohols, amino acids, peptides, and/or aromatics), an end electronacceptor (such as oxygen, nitrate, nitrite, nitrous oxide, sulfate,and/or ferric iron), and “feed nutrients” (sulfate, sulfide, phosphate,and a nitrogen source, such as nitrate, ammonia, and/or urea). Theorganic source supplies energy and carbon molecules for anabolism. Theend electron acceptor functions as the molecule that is reduced duringenergy generation. Feed nutrients are needed as building blocks formacromolecule synthesis of cellular constituents such as amino acids,deoxyribonucleotides, ribonucleotides, and phospholipids. Preferably,more than one nutrient is present in the feed nutrients. In a specificembodiment of the invention, the feed nutrients comprise free ammonia, asulfate, and potassium phosphate or ammonium phosphate as nutrients.

[0046] The term “end electron acceptor” will mean a molecule that isreduced as a result of microbial respiration. Examples include, but arenot limited to, oxygen, nitrate, nitrite, nitrous oxide, ferric oxide,and sulfate.

[0047] The term “nitrate-N” will mean the amount of nitrogen present permole of nitrate. The conversion factor is 4.5 g nitrate/g nitrogen.Therefore, 20 mg/L nitrate-N is equal to 90 mg/L nitrate.

[0048] The term “mixed liquor suspended solids” or “MLSS” refers to thedry weight of the biomass in the system and is measured by drying tocompletion a known volume from the bioreactor at 105° C.

[0049] The term “mixed liquor volatile suspended solids” or “MLVSS”refers to the weight of volatile suspended solids in the system and isdetermined by heating the dried sample at 550° C. for 30 min, followedby cooling to room temperature in a dessicator, and measuring theweight. “MLVSS” is calculated by subtracting the residual weight of thesample following the 550° C. heating step from “MLSS” weight.

[0050] The terms “activated sludge bacteria”, “activated sludgemicroorganisms” or “sludge” will refer to autotrophic, heterotrophic andfacultative microorganisms that are typically found in wastewatersystems and possess the enzymatic machinery to degrade compounds foundin carbon influx.

[0051] Activated Sludge Environment

[0052] The present invention provides an activated sludge environmentcomprising a number of components including a carbon source, a varietyof autotrophic, heterotrophic, and/or facultative bacteria, and feednutrients required for the growth and biocatalytic health of thebiomass. Preferably, all three types (autotrophic, heterotrophic andfacultative) of bacteria are present.

[0053] The term “autotrophic bacterium” or “autotrophic bacteria” willmean an organism or organisms capable of growing on inorganic nutrientsand using carbon dioxide as its sole carbon source.

[0054] The term “heterotrophic bacterium” or “heterotrophic bacteria”will mean an organism or organisms that requires one or more organicnutrients, including an organic carbon source for growth, e.g., therequirement of glucose by Escherichia coli for growth.

[0055] The term “facultative bacterium” or “facultative bacteria” willmean an organism or organisms that can switch easily from one growthphysiology to another. For example, bacteria that can switch to nitrateas an electron acceptor in the absence of oxygen.

[0056] The types of bacteria that may be present in the activated sludgeenvironment include, but are not limited to, Proteobacteria, aphysiologically diverse group of microorganisms that represents fivesubdivisons (α, β, γ, ε, δ), (Madigan et al. Biology of Microorganisms,8^(th) ed. Prentice Hall Upper Saddle River, NJ 1997). Specific examplesof bacterial genera that are expected to work or be involved in theactivated sludge environment include, but are not limited to,Paracoccus, Rhodococcus, Pseudomonas, Alcaligenes, Acinetobacter,Sphingamonas, Azoarcus, and Burkholderia.

[0057] Internal Storage Molecules:

[0058] The production of an internal storage molecule is used within themethods of the present invention to monitor and control bioreactorhealth, to predict and assess bioreactor catalytic efficiency, and tomaintain an activated sludge environment. This internal storage moleculemay be a class of molecules, collectively termed polyhydroxyalkanoates(PHA), or glycogen, or the like. Both PHA and glycogen have beenextensively studied as energy-storage compounds (see Dawes and Senior,1973. Adv. Microb. Physiol. 10:135-266) but their regulation is notclearly understood. In a preferred embodiment of the present invention,this internal storage molecule is polyhydroxyalkanoates (PHA).

[0059] Polyhydroxyalkanoates:

[0060] The terms “polyhydroxyalkanoates” or “PHA” are used herein asgeneric terms for a class of molecules that are primarily linear,head-to-tail polyesters composed of 3-hydroxy fatty acid monomers thataccumulate in microbes as carbon and energy storage molecules (seeMadison and Huisman, 1999. Microbiol. Mol. Biol. Rev., 63:21-53; andByrom, 1994. In: Mobley, ed. Plastics from Microbes, Hanser Publishers,New York, pp. 5-33 for reviews). These polymers are generallysynthesized in a broad range of bacteria when the cells have adequatecarbon supplies but are limited for another nutrient, such as nitrogen,phosphate, or oxygen.

[0061] The terms “polyhydroxyalkanoates” or “PHA” refer to a class ofcompounds of the general formula:

[0062] where R=CH₃ or CH₃(CH₂)m, where m=1 to 10, and n=4,000 to 20,000(Mobley, D, editor. Plastics From Microbes. Hanser Publishers, New York,1994).

[0063] The most commonly occurring PHA molecules are polyhydroxybutyrate (PH B) and polyhyd roxyvalerate/polyhyd roxybutyrateco-polymer that results from a condensation of two molecules of acetylCoA to form 3-hydroxybutyryl CoA or acetyl CoA with propionyl CoA toform 3-hydroxyvaleryl CoA. These activated molecules are thenincorporated into the polymer.

[0064] In addition to the 3-hydroxyalkonoates described above, somebacteria have the ability to incorporate longer chained 3-hydroxy acids(carbon length up to C₁₄) into the backbone of the polymer (see Byrom,1994 supra). A list of monomers and functional groups that have beenfound in microbial PHA is disclosed in Table 2.5 of Byrom (1994).Typically, the composition of the resulting PHA depends upon the growthsubstrate used (Madison and Huisman, 1999, supra). While the lociencoding PHA synthesis genes have been characterized for at least 18different species, PHA biosynthesis and its coordinated gene expressionin response to environmental conditions remains unclear and represents acomplex area under current investigation.

[0065] Quantitation of PHA Levels:

[0066] PHA levels within a sample can be determined or measured forexample, by removing an aliquot of the biomass from the reactor anddetermining its dry weight. Briefly, the biomass is lysed by addition of0.1 mL of concentrated hydrochloric acid and 0.4 mL of n-propanol, andheat treatment at 100° C. for 2 hours. During this lysis step,polyhydroxyalkonoates are hydrolyzed and esterified. The esterifiedcompounds can then be quantitated by gas chromatography (GC) asdescribed by Riis and Mai, (Riis and Mai (1988) supra) and the dryweight percent PHA calculated.

[0067] PHA levels can also be determined using an automated fluorescencebased PHA method based upon that described by Ostle and Holt, 1982(Applied and Environmental Microbiology, 44:23) and modified asdescribed below. Briefly, one milliliter of biomass is transferred fromthe bioreactor to a chamber into which is added 1 mL of Nile Blue (1%).The mixture is incubated at 55° C. for 15 min. The sample is washed withdeionized water (or some suitably “clean” non-fluorescent liquid) toremove the excess Nile Blue dye followed by an 8% acetic acid wash. Thestained biomass is transferred to a cuvette/flow cell in a fluorescencespectrophotometer with the excitation wavelength set to 362 nm. Theamount of PHA present in the biomass is determined by comparison to astandard curve generated using either neat PHA polymer or biomasscontaining PHA that has been quantitated using the GC-FID (flameionization detector) method.

[0068] High levels of PHA in the biomass indicate that carbon flow inthe cell has been directed toward PHA formation and is an indicator ofreduced bioreactor health. PHA production and denitrification competefor reduced forms of nicatinamide deoxyribose [NAD(P)H]. Therefore, PHAformation can be viewed as a non-productive side reaction reducing theperformance of the system for the desired reaction, i.e. reduction ofnitrate.

[0069] Specifically, Applicants have determined that when PHAaccumulates to high levels in the biomass (about 15 to about 20% dryweight), then utilization of exogenously supplied carbon (methanol andorganic acids) slows. Appropriate adjustment of the feed nutrients tothe bioreactor can then be made to return the bioreactor to an efficientoperating condition. Applicants' observation that high levels of PHA inthe biomass correlate to reduced denitrification activity can be used tomore rapidly assess the productivity of anoxic wastewater reactors.Since the build up of PHA in bacteria is primarily driven by nutrientlimitation (sulfate, sulfide, phosphate, and a nitrogen source, such asnitrate, ammonia, and/or urea), the flux of carbon into PHA can beeasily controlled by monitoring key nutrient levels in the effluent.

[0070] Wastewater Process Monitoring and Adjustments in Response toIncreased PHA Level

[0071] The overall goal of wastewater systems is to reliably andefficiently treat inlet streams (municipal or industrial) to convertorganic carbon to carbon dioxide, and in some cases, organic andinorganic nitrogen to gaseous end products. The general strategy is touse monitoring of nutrient and carbon consumption along with PHA levelsin the biomass as a gauge of system productivity to ensure performanceof the bioreactor.

[0072] An indication that the biocatalytic efficiency of the wastewatertreatment process is impaired is seen when the polyhydroxyalkanoatesconcentration is greater than about 15 to about 20 dry weight percent ofthe biomass. Appropriate adjustment of the feed nutrients to thebioreactor can then be made to return the bioreactor to an efficientoperating condition. Preferably, the feed nutrients are adjustedaccordingly in the activated sludge environment when the PHAconcentration is from about 10 to about 20 dry weight percent of thebiomass. More preferably, the feed nutrients are adjusted accordingly inthe activated sludge environment when the PHA concentration is fromabout 10 to about 15 dry weight percent of the biomass.

[0073] Maintaining Viable Cultures in a Bioreactor During Reduced orAbsent Carbon Influx

[0074] The present invention also provides the means to maintain anactivated sludge system in the absence of carbon influx. Specifically,applicants have developed a method for maintaining biological activityin the absence of carbon influx to the bioreactor. This method isparticularly useful during periods of reduced wastewater production,specifically during scheduled shut downs of the manufacturing process,which normally supplies the carbon feed to the bioreactor. Currently,the only recourse to maintain viable biomass within the reactor is tosupply it with purchased carbon sources, such as corn steep liquor,molasses, or another cheap carbon source. By understanding themechanisms that promote PHA accumulation and providing methods to easilymonitor and control the amount of PHA present in the biomass, thesepolymers can be used as internally supplied carbon sources. The abilityof most natural bacteria to break down stored carbon for use as a carbonand energy source during periods of starvation is exploited in thismethod to maintain biological activity in the wastewater bioreactorwithout the need to purchase carbon sources.

[0075] Prior to a scheduled shut down, nutrient limitations can be usedto shift the microbial physiology to PHA synthesis. Applicants haveshown in Example 3 below that internal PHA is able to function as acarbon source for the biomass during periods of process waste outages.PHA formation can be stimulated by limiting the amount of nitrogen,phosphorous, and/or sulfur sources that is supplied to the biomass. In aspecific embodiment, PHA formation can be stimulated by limiting theamount of nitrogen that is supplied to the biomass. The nitrogen sourcemay include but is not limited to ammonia, nitrate, and/or urea.Preferably, nutrient limitation of the biomass will begin from about 8weeks before to about 2 weeks before shut down. More preferably,nutrient limitation of the biomass will begin from about 8 weeks beforeto about 6 weeks before shut down. Even more preferably, nutrientlimitation of the biomass will begin at about 8 weeks before shut down.The biologically required amount of nutrients can be determined from thegrowth yield data, carbon load to the reactor and the amount of eachnutrient present in biomass (Wastewater Engineering: Treatment,Disposal, and Reuse. Metcalf and Eddy, Inc. 3^(rd) Edition. McGraw-Hill,Inc., New York, 1991).

[0076] During the shut down period, the level of PHA in the systemshould be followed to insure that the biomass does not starve.Preferably, the level of PHA in the system will be at least than about1% to about 5% of the MLSS. More preferably, the level of PHA in thesystem will be at least than about 3% to about 5% of the MLSS. Even morepreferably, the level of PHA in the system will be at least than about5% of the MLSS. If the PHA level approaches about 1% to 2% of the MLSS,then purchased carbon sources can be supplied to keep the bioreactorviable until wastewater carbon influx is resumed.

EXAMPLES

[0077] The present invention is further defined in the followingnon-limiting Examples. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various uses and conditions.

[0078] General Methods

[0079] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,DC. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989).

[0080] Methods for analysis and determination of components present inwastewater bioreactors can be found in Eaton et al. (Standard Methodsfor the Examination of Water and Wastewater, 19^(th) edition, AmericanPublic Health Association, Washington, D.C., 1995).

[0081] All reagents, restriction enzymes and materials used for thegrowth and maintenance of bacterial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.) unless otherwise specified.

[0082] The meaning of abbreviations is as follows: “h” means hour(s),“min” means minute(s), “sec” means second(s), “d” means day(s), “mg”means milligram, “g” means gram, “uL” means microliters, “mL” meansmilliliters, “L” means liters, “uM” means micromolar, “mM” meansmillimolar, “M” means molar, and “ppm” means parts per million.

[0083] Bioreactor Operation:

[0084] A modified Eckenfelder reactor was operated in continuous mode.The modifications included a water jacket for temperature control and ahead plate. The reactor volume was 280 mL and contained an internalsettling zone of 50 mL for a total volume of 330 mL, the temperature wasmaintained at 35° C. and the pH controlled to 7.50+/−0.15. The pH wasmaintained by addition of 0.1 M NaOH. The flow rate was set to 0.12mL/min establishing a hydrolic residence time of 1.9 days. The sludgeresidence time (sludge age) was 20 days. The sludge age was controlledthrough daily wasting of biomass from the system. Biomass wasting wasperformed by mixing the contents of the settling and reaction zones andremoving {fraction (1/20)}th of the reactor volume (20 mL/d). The massof bacteria present in the reactor was determined daily as follows:three milliliters of biomass was filter through a dried, preweighed 1.2μM glass filter (Gelman, Ann Arbor, Mich.), the biomass was dried for 1hour at 105° C. (dry weight) and combusted at 550° C. for 20 minutes(ashe weight). The oxidation reduction potential (ORP), pH, andtemperature were continuously logged to a computer through a dataacquisition system (10 Tech multiscan with DuPont Scan 1200 V1.3.0software).

[0085] Media Formulation:

[0086] The feed to the reactor was prepared in sterile deionized water.Nitric acid was added to bring the concentration of nitrate to 13.5 g/L.Two compositions of organics were used: 1) methanol and 2) a combinationof methanol, valeric acid and butyric acid. The methanol feed contained9.0 g/L and the mixture contained 4.5 g/L methanol, 1.79 g/l valericacid and 1.96 g/L butyric acid. The pH was adjusted in both feedcompositions to approximately 1.3 by the addition of 2 mL of 50% sodiumhydroxide.

[0087] Nutrient Addition:

[0088] The reactor was batch fed (75 μL/d) of the following nutrientsolutions: Solution 1: comprising Na₂SO4, 82.0 g/L; KH₂PO₄, 65.6 g/L;H₃BO₃, 1.1 g/L; NaMoO₄, and 0.5 g/L, NiCl 6H₂O; Solution 2 comprisingF₃C; 4H₂O, 13.5 g/L); Solution 3 (MnCl 4H₂O, 7.0 g/L; CaCl₂ 2H₂O, 63.3g/L; MgCl₂ 6H₂O, 110.3 g/L; CuCl₂ 2H₂O, 0.5 g/L; CoCl₂ 6H₂O, 0.8 g/L).During defined periods, ammonia was also added to the system to a finalconcentration that ranged from about 99 to about 396 mg/L. The amountsof phosphate, sulfate and ammonia were determined by ion chromatography(see below).

[0089] Analytical Techniques

[0090] COD Analysis:

[0091] The chemical oxygen demand (COD) of the feed and in thebioreactor was measured using Hach COD (Hach Corp., Loveland Colo.)vials following the manufacturer's protocol.

[0092] Methanol Analysis:

[0093] The concentration of methanol in the reactor was determined byGC/FID as described above (Riis and Mai (1988) supra). Briefly, biomasswas removed from the reactor and centrifuged at 13,000 rpm in amicrofuge (Heraeus Instruments, USA) at4° C. The supernatant wasfiltered through a 0.2 micron filter (Gelman). An HP 6890 GC HewletPackard instrument containing a HP-5 capillary column was operated usingthe following parameters: inlet temperature of 250° C., a helium carriergas flow rate at 1.5 mL/min, a column temperature initially set at 60°C. and ramped to 250° C. at 25° C./min, an FID temperature of 250° C.and a hydrogen gas flow rate of 40 mL/min, an air flow rate of 450mL/min, and a helium flow rate of 45 mL/min. An injection volume of 2 uLwas used with a 20:1 split at the inlet. Cyclopentanone was used as theinternal standard and a linear calibration curve for methanolconcentrations ranging from 0 to 1000 mg/l (r² 0.99) was used todetermine reactor methanol concentration.

[0094] Anion Analysis:

[0095] The concentrations of valeric acid, butyric acid, nitrate, andnitrite were determined by Ion Chromotography using a Dionex IC System.Anions were analyzed using the Dionex ion chromatography isocraticmethod for anion analysis. Briefly, AS11-HC analytical columns withAS11-HC guard were used with a pump flow rate of 1.5 mL/min. TheDetector was conductivity and the eluent was 24 mM NaOH. Sample runtimewas 16 minutes using an injection volume of 25 μL. The primary anionsanalyzed were chloride, nitrite, sulfate, nitrate, and phosphate.External standards were used to generate standard curves. Standardconcentrations of 0 mg/L, 1 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, and 50 mg/Lwith r²>0.99 for each component were used to generate the standardcurves. Samples were taken from the reactor at different times andcentrifuged in 1.5 mL micro-centrifuge tubes for 5 min at 13,000 rpm.The supernatant was diluted 1:5 with deionized water and placed on theautosampler for analysis.

[0096] Cation Analysis:

[0097] Cations were analyzed using the Dionex ion chromatographyisocratic method for cation analysis. Briefly, CS12A analytical columnswith CS12G guard were used with a pump flow rate of 1.0 mL/min. Thedetector was conductivity and the eluent was 22 mN H₂SO₄. Sample runtime was 16 min using an injection volume of 15 μL. The primary cationsanalyzed were sodium, ammonium, potassium, magnesium, and calcium.External standards were used to generate a standard curve. Standardconcentrations of 0 mg/L, 5 mg/L, 10 mg/L and 25 mg/L, with r²>0.99 foreach component were used to generate the standard curves. Samples weretaken from the reactor at different times and centrifuged in 1.5 mLmicro centrifuge tubes for 5 min at 13,000 rpm. One mL of the sample'ssupernatant was the placed into a GC screw top vial containing 10 μL ofH₂SO₄ and either placed in the refrigerator at 4° C. or diluted 1:5 withdeionized water and placed on the autosampler for analysis.

[0098] Nitrous Oxide Analysis:

[0099] The dissolved nitrous oxide concentration was determined by thefollowing method. Briefly, a 1 mL reactor sample was placed into a 2.0mL crimp cap vial and the sample was acidifed by adding 10 μL ofconcentrated HCL to stop biological activity. Samples were stored at 4°C. until analyzed. Samples were heated overnight at 80° C. to establishequilibrium between the dissolved and gaseous nitrous oxide. Nitrousoxide levels were determined by GC/ECD (HP instruments) using a Supelco80/100 Porapak Qss column. The inlet and column temperatures were 30° C.and the total gas flow rate was 30 mL/min (5% methane, 95% Argon). Theinjection was 100 μL of the vial head space and the GC was operated insplitless mode. Nitrous oxide concentration was determined from anexternal standard curve made using gas standards. 0, 10, and 100 ppm N₂0(Roberts Gas, Co.).

[0100] PHA Concentration Determination:

[0101] Biomass was removed from the reactor and transferred to apreweighed 7 mL vial and centifuged at 4000 rpm for 10 min in a SorvalSS34 rotor at 4° C. The supernatant was removed and the biomass pelletis dried at 105° C. overnight. The dry weight of the biomass wasdetermined. The mass of bacteria present in the reactor was determineddaily as follows: three milliliters of biomass was filter through adried, preweighed 1.2 μM glass filter (Gelman), the biomass was driedfor 1 hr at 105° C. (dry weight) and combusted at 550° C. for 20 minutes(ashe weight).

[0102] The common PHB method is derived from the method developed byRiis and Mai (Riis and Mai (1988) supra).

[0103] The method is based on the hydrolysis and transesterification ofPHB with propanol and hydrochloric acid to hydroxybutryric acid propylester. Benzoic acid is used as the internal standard. Briefly, thismethod involves adding 1 mL of Dichloroethane (DCE), 200 μL of Benzoicacid stock solution (2.0 g Benzoic acid in 50 mL of 1-propanol), and 1mL of a Propanol-HCl stock solution (1 volume concentrated hydrochloricacid and 4 volumes 1-propanol) to the vial containing the dry biomasssample. The re-suspended sample is then reacted in a heat block for 2hours at 100° C. with periodic shaking. The sample is then cooled toroom temperature and the reaction is quenched by adding 2 mL ofdeionized water. Five μL of the organic phase (lower phase) is removedand injected into an HP 6890 GC equipped with a HP-5 5% PhenylMethyl-Siloxane capillary column. The GC analysis parameters used were agas flow rate of 1.5 mL/min, injection made with the inlet-split mode,with a split ratio=100:1, an inlet temperature of 180° C. with helium asthe carrier gas. The column temperature was initially 100° C. and at theend of the method was 225° C. using a 25° C./min ramp rate. The columnwas held at 100° C. for 0.5 min, and the total run time was 7.0 min. Thedetector was a flame ionization detector (FID) operated at 275° C. Thegas flow rate was 40 mL/min (Hydrogen) and the air flow rate was 450mL/min with a Helium make up flow of 50 mL/min.

[0104] PHA Calibration Standards Preparation Procedure:

[0105] The PHA concentration is determined by comparison to standardcontaining known amounts of PHBA and PHVA (Sigma Chemical company). A 20g/L PHB/PHV stock solution was prepared by dissolving 100 mg of PHB/PHVin 5 mL dichloroethane (DCE) and heating at 100° C. The vesselcomprising this stock solution should be a calibrated sealed vial. Afterthe PHB/PHV goes into solution, it was cooled to room temperature andDCE was added to restore the original volume. This 20 g/L PHB/PHV stocksolution and dilutions prepared from it can be used to detect a range ofreactor biomass PHA concentrations from 0 mg/ml to about 16.4 mg/ml. Themaximum amount of biomass used was approximately 15 mg, therefore, theamount of PHA in the reactor biomass was within the range of the PHAconcentrations provided by the standards.

[0106] PHA concentration can also be determined using the automatedfluorescence method based upon Ostle and Holt (1982) and modified asdescribed above in the Detailed Description of the Invention.

[0107] Determination of Denitrification Potential:

[0108] Denitrification potential, the rate of reduction of nitrate tonitrogen per unit of biomass, was determined using biomass from ananoxic bioreactor. The biomass was removed from the reactor andtransferred to a centrifuge tube, pelleted and washed with a phosphatebuffer solution (pH 7.5, 50 mM), and resuspended in an S12 mineral saltsmedia (10 mM NH₄SO₄; 50 mM KPO₄, pH 7.0; 2 mM mgCl₂; 0.7 mM CaCl₂; 0.05mM MnCl₂; 0.001 mM FeCl₃; 0.001 mM ZnCl₃; 1.72 μM CuSO₄; 2.53 μM COCl₂;and 2.42 μM NaMoO₂) and sparged with argon to remove oxygen. The vialscontained between 400 to 500 mg/L of reactor biomass. Various carbonmixes and 20 mg/L nitrate-N (wherein 20 mg/L nitrate-N multiplied by 4.5g NO₃/gN=90 mg/L nitrate) were added into anaerobic vials to initiatethe reaction. The carbon sources used consisted of the feed composition(50/50 methanol and organic acids), methanol, organic acids (valeric andbutyric) and a no carbon control. The reaction using the feedcomposition contained 33 mg/L methanol, 12.3 mg/L valeric acid and 13.6mg/L butyric acid. The methanol reaction contained 66 mg/L methanol andthe organic acids reaction contained 24.5 mg/L valeric acid and 27.2mg/L butyric acid. The rates of nitrate removal and carbon consumptionwere measured as a function of time using the ion chromatography methoddescribed above. The studies were performed at 350C and pH 7.5, theoperating conditions of the anoxic reactor.

Example 1 Correlation Between PHA and Denitrification in IsolatedCultures

[0109] This example describes the impact of feed composition ondenitrification rates as determined in a continuous anoxic bioreactor.The first operating condition comprised a feed composition comprising90% methanol, 5% adipic acid, 2.5% cyclopentanone, and 2.5%hexamethylenediamine, referred to herein as “methanol rich feed”. Thesystem was operated in this regime for several sludge ages, i.e., equalto the amount of time needed to replace all of the biomass present inthe reactor, so that the operating performance and systemcharacteristics using the methanol rich feed could be determined.Following this period, the system was switched to a feed compositioncomprising 50% methanol, 25% butyric acid and 25% valeric acid, referredto as “organic acid rich feed” (Arrow 1 on FIGS. 1 and 2). The systemwas maintained on this feed for several sludge ages and characterized atthe macroscopic level using measurements of carbon, nitrate/nitrite, andbiomass (MLVSS and MLSS). At various times during this operating period,batch studies were used to measure the denitrification uptake rate (NUR)on various carbon sources. The carbon sources used consisted of the feedcomposition (50/50 methanol and organic acids), methanol, organic acids(valeric and butyric acids), and a no carbon control.

[0110] The macroscopic performance of the methanol rich feed system isshown in FIG. 1 and Table 1. TABLE 1 Reactor Performance without AmmoniaAddition Feed NO₃ Load Reactor NO₃ Effluent NO₃ Day mg/day mg/L mg/L 12246.0 8.0000 0.0000 2 2184.0 0.0000 0.0000 5 2278.0 8.0000 8.0000 82278.0 ND ND 12 1917.0 ND ND 25 1874.0 0.0000 13.000 32 1642.0 0.00000.0000 39 2421.0 0.0000 0.0000 43 2421.0 ND 0.0000 46 2328.0 0.00000.0000 53 2384.0 0.0000 17.000 60 2233.0 0.0000 0.0000 62 2213.0 183.00ND 64 2213.0 4.0000 ND 67 2182.0 0.0000 0.0000 71 2223.0 0.0000 ND 74 ND0.0000 ND 75 2286.0 491.00 8.0000 76 ND 0.0000 ND 81 2178.0 0.00000.0000 82 2050.0 ND ND 83 2060.0 40.000 0.0000 88 2203.0 0.0000 0.000090 1851.0 104.00 0.0000 91 1194.0 0.0000 0.0000 92 1191.0 8.0000 8.000093 0.0000 ND ND 95 2401.0 ND 0.0000 96 ND 83.000 ND 97 2401.0 ND ND 98ND 0.0000 ND 99 2286.0 0.0000 ND 101 ND 0.0000 ND 102 2300.0 8.00008.0000 104 2181.0 8.0000 0.0000 106 2170.0 0.0000 0.0000 109 2250.08.0000 0.0000 111 2151.0 46.000 8.0000 113 2276.0 29.000 0.0000 1162294.0 103.00 0.0000 118 2143.0 94.000 8.0000 120 2149.0 60.000 0.0000123 2222.0 0.0000 0.0000 125 2447.0 0.0000 0.0000 127 2438.0 866.000.0000 130 1865.0 0.0000 0.0000 132 1782.0 81.000 0.0000 133 ND 346.00ND 134 1747.0 500.00 0.0000 137 2038.0 129.00 0.0000 139 ND 1641.0 ND140 1905.0 865.00 38.000 143 1848.0 81.000 0.0000 146 ND 433.00 ND 147ND ND ND 148 1118.0 1224.0 0.0000 151 1476.0 0.0000 0.0000 153 1776.0170.00 0.0000 154 21.000 ND ND 155 16.000 14.000 0.0000 158 11.00016.000 0.0000 160 1065.0 19.000 12.000 165 ND 180.00 0.0000 166 236.00264.00 0.0000 167 ND 0.0000 ND 168 102.00 74.000 ND 169 114.00 37.0000.0000 172 117.00 0.0000 0.0000 173 155.00 144.00 0.0000 174 554.0017.000 ND 175 1044.0 0.0000 0.0000

[0111] Operation on the methanol rich feed composition resulted inrobust performance as characterized by no nitrite or low residualnitrate levels in the effluent. Efficiency of denitrification droppeddramatically following the shift to the organic acid rich feedcomposition (Arrow 1, FIG. 1). The nitrate load to the system wasreduced to zero to simulate shut down two times (Arrows 2 and 3, FIG. 1)and residual carbon and nitrate were flushed from the reactor using a 5mM phosphate buffer, pH 7.5.

[0112] The impact of these operating conditions on NUR using variouscarbon sources and the corresponding reactor PHA levels is shown in FIG.2. Operation with the methanol rich feed resulted in consistent NURvalues in the range 3.35×10⁻⁴+/−0.2×10⁻⁴ mg NO₃-N/mg MLVSS-min withmethanol as the carbon source (FIG. 2A). This denitrification rate issufficient to ensure complete reduction of the feed nitrate. Following ashift to the organic acid rich feed (Arrow 1 on FIG. 2A), a significantreduction in the rate of denitrification was observed. The NUR withmethanol as the carbon source decreased 10 fold within 21 days followingthe feed shift (FIG. 2B). Also during this period, the endogenous rateincreased by 3 fold as determined in the no carbon control shown in FIG.2C. The increase in the endogenous rate indicates that the biomass wasusing an internally stored carbon source. However, the NUR with the feedcomposition was reduced (approximately 3 fold) during in the same timeframe (21 days) as shown in FIG. 2A. These data indicate a generalreduction in the specific NUR as a function of time following the switchfrom methanol rich feed to a feed comprising both methanol and organicacids and referred to as the organic acid rich feed. As mentioned above,the reactor was not able to continually metabolize the nitrate andcarbon at the rate at which it was being fed. The feed was then switchedto a phosphate buffer (pH 7.5) for 24 hours to flush the residual carbonand nitrate from the reactor (Arrow 2 on FIG. 2A). The system wasrestarted on the organic acid rich feed. The NUR on the organic acidrich feed composition was reduced by approximately 2 fold with the lossof denitrification performance observed within a similar time frame,approximately 21 days (see FIG. 2A) as compared to the first period ofoperation. The NUR with methanol as the carbon source decreased by afactor of 10 (FIG. 2B). Also consistent with the first period ofoperation, the endogenous NUR of the second operation increased by afactor of 1.5 (FIG. 2C).

[0113] The loss of nitrate reduction performance shown in FIG. 1(approximately day 95) was reproducible. This behavior was repeated byoperating the reactor under the same conditions that resulted in loss ofnitrate metabolism. These data are shown in FIG. 1 from 98 to 155 days(between Arrows 2 and 3). The macroscopic characteristics of the systemwere the same, i.e. reduced nitrate reduction rates and increased levelsof PHA in the biomass as shown in FIG. 2. Note the arrow numbers inFIGS. 1 and 2 refer to the same operating periods. These data indicatethat the loss of denitrification (nitrate reduction) performance isreproducible and driven by a decreased ability of the biomass to use theexogenously supplied carbon to drive denitrification as indicated by anincreased NUR in the no carbon added sample (FIG. 2C). Applicants haveherein determined that the internal carbon source present in the biomassis PHA (see FIG. 2D).

[0114] The correlation between NUR and PHA content in the biomass isshown in FIG. 3 and Table 2. The batch NUR measured with the organicacid rich feed composition was reduced by a factor of 3-4 at PHA levelsabove 15-20%. The data presented in FIG. 3 and Table 2 are a compositeof all of the data collected during the various operating conditions.TABLE 2 Percent maximum specific nitrate uptake rates and percent PHA %Maximum % Maximum SNUR SNUR % MeOH Feed PHA 100.0 143.0 0.0 125.0 100.01.0 75.0 84.0 1.0 46.0 49.0 2.0 94.0 93.0 6.0 63.0 92.0 6.0 66.0 86.07.0 64.0 96.0 8.0 58.0 84.0 11.0 64.0 55.0 15.0 100.0 63.0 17.0 80.086.0 18.0 4.0 17.0 24.0 3.0 23.0 24.0 2.0 16.0 25.0 7.0 28.0 26.0 7.030.0 28.0 7.0 20.0 28.0 9.0 22.0 30.0

[0115] Although, some of the data fall outside of the general trend, themajority is consistent with the observation that increased PHA levels inthe biomass resulted in reduced nitrate uptake rates. With methanol asthe carbon source, the NUR was reduced approximately by an order ofmagnitude at PHA levels above 20%.

[0116] The impact of PHA levels on system performance can be determinedby comparing the system's nitrate load, which is equal to [feed nitrate(mg NO₃-N/L)]×[flow rate (L/min)], to the denitrification capacity inthe reactor. The system capacity can be estimated by multiplying themaximum NUR by a PHA inhibition term and the total amount of biomass inthe system using Equation 2.

System capacity=[Maximum nitrate rate (mg nitrate-N/mgMLVSS-min)−reduction due to PHA]×[Total biomass in reactor (mgMLVSS)].  EQUATION 2:

[0117] The term for the reduction in NUR caused by PHA is determined bylinear regression analysis of the data shown in FIG. 3 as shown inEquation 3.

NUR reduction=(1.5×10⁻⁵ mg nitrate-N/mgMLVSS-min-%PHA)×(%PHA).  EQUATION 3:

[0118] The system capacity must be greater than or equal to the nitrateload to the system for complete denitrification to occur. Therefore,Applicants' invention provides one of ordinary skill in the art theability to monitor PHA levels within the biomass of a wastewaterbioreactor and predict the denitrification capacity in the reactor todetermine system performance. Appropriate adjustments to the influx,such as nutrient control, can be made similar to that described inExample 2 below to maintain efficient system performance.

Example 2 Effect of the Addition of Various Feed Nutrients on PHA Levels

[0119] Example 2 demonstrates that denitrification performance isimpacted by PHA level in the biomass. Specifically, an increase in PHAcontent corresponded to a diminished ability of the biomass to usemethanol as a carbon source to drive denitrification (see FIGS. 2 and 3and Table 2). Accumulation of PHA is a physiological response to excesscarbon and nutrient starvation, and can be minimized by the addition ofmacro- and micronutrients such as ammonia, sulfate, and phosphate. Areactor feed composition was chosen that contained excess ammonia (1.5times the physiologically required amount) to minimize PHA accumulationand promote reliable denitrification performance.

[0120] To test the ability of ammonia to control PHA levels in thebiomass, the organic acid rich feed composition described in Example 1was used since PHA levels were higher with this feed composition. Themacroscopic performance of the system is shown in FIG. 4. Duringoperation with excess ammonia, the system performance was very stableand reliable and no nitrate leaks were observed. Performance under thesereactor conditions contrasts significantly to operation without theaddition of ammonia as described in Example 1 (see FIG. 1).

[0121] NUR experiments were conducted by using biomass from the anoxicbioreactor as described above in Example 1. The nitrate uptake rateusing various carbon sources and the corresponding reactor PHA levelsare shown in FIG. 5. The NUR with the organic acid rich feed compositionas the carbon source with ammonia shown in FIG. 5A resulted in moreconsistent nitrate removal rates, ranging from 2.82×10⁻⁴ to 6.75×10⁻⁴ mgnitrate-N/mg MLVSS-min. The average feed NUR (organic acid rich feedcomposition) was approximately 30% higher with ammonia addition thanwithout ammonia addition (compare FIGS. 5A to 2A, respectively). Therate of nitrate removal with methanol as the carbon source ranged from1.49×10⁻⁴ to 3.24×10⁻⁴ mg nitrate-N/mg MLVSS-min as shown in FIG. 5B.These rates are comparable to the maximum NUR observed during theprevious operating regimes (methanol rich feed and organic acid richfeed, both without ammonia addition, FIGS. 1 and 2). The relativelyuniform NUR during this period (FIG. 5B) is a sharp contrast to thehighly variable NUR rate (methanol as the carbon source) observed duringoperation with organic acid rich feed without nutrient control (FIG.2B).

[0122] The concentration of PHA in the biomass as a function ofoperating condition is shown in FIG. 5D. The levels of PHA wereapproximately two fold higher in the absence of ammonia addition(nutrient control, see FIG. 2D) as compared to operation with nutrientcontrol (FIG. 5D). Without nutrient control, a large fraction (15 to38%) of the biocatalyst (biomass) is inert polyester (PHA) (FIG. 2D). Incontrast, nutrient addition controls the amount of PHA to about 10% orless (FIG. 5D). These results are consistent with the low endogenous NURobserved in the no carbon control reactions as shown in FIG. 5C.

[0123] Addition of excess ammonia resulted in reduced levels of PHA inthe biomass and consistent nitrate uptake rates. The nitrate utilizationrate was approximately 30% higher than the maximum NUR observed in thetwo previous operating regimes (methanol rich, and organic acid rich,both without ammonia). These results highlight the impact of operatingconditions on the performance of an anoxic system and the need tomonitor both biomass composition and levels in wastewater bioreactors.Addition of excess ammonia resulted in stable and reliabledenitrification performance and reduced levels of PHA in the biomass.

[0124] Therefore, monitoring of PHA and nutrient levels in wastewatertreatment systems can be used to gauge the denitrification potential andas a general measure of system health. An indication that thebiocatalytic efficiency of the wastewater treatment process is impairedis seen when the polyhydroxyalkanoates concentration is greater thanabout 15 to about 20 dry weight percent of the biomass. Appropriateadjustment of the feed nutrients to the bioreactor can then be made toreturn the bioreactor to an efficient operating condition. Preferably,the feed nutrients are adjusted accordingly in the activated sludgeenvironment when the PHA concentration is from about 10 to about 20 dryweight percent of the biomass. More preferably, the feed nutrients areadjusted accordingly in the activated sludge environment when the PHAconcentration is from about 10 to about 15 dry weight percent of thebiomass. Adjustments made to the feed nutrients (i.e., addition ofexcess ammonia, nitrate, urea, phosphorous sources, sulfur sources, andthe like) are effective to achieve optimum health and denitrificationability of the bioreactor.

Example 3 An Activated Sludge System in the Absence Of Carbon Influx

[0125] This example describes a method for maintaining biologicalactivity during periods of reduced or absent carbon influx to thebioreactor. This method is useful during periods of scheduled shut downsof the manufacturing process, which normally supplies the carbon feed tothe bioreactor, or any other time of significantly reduced carboninflux. Currently, the only recourse to maintain viable bioreactorcultures during a shut-down period is to supply purchased carbon sourcessuch as corn steep liquor, molasses, or another cheap carbon source tothe bioreactor. Applicants have identified the conditions under whichPHA accumulation can be promoted and provide methods to easily monitorand control the amount of PHA present in the biomass. Since thesepolymers can be used as internally supplied carbon sources, PHAaccumulation can be achieved prior to the scheduled shut down or lowcarbon influx period. The ability of most natural bacteria to break downthe stored PHA for use as a carbon and energy source during periods ofstarvation can then be exploited in a method to maintain biologicalactivity in the wastewater bioreactor without the need to purchasecarbon sources.

[0126] Applicants have demonstrated herein that nutrient limitations canbe used to shift the microbial physiology to PHA synthesis prior to ascheduled shut down. Specifically, PHA formation can be stimulated bylimiting the amount of nitrogen, phosphate, or sulfate that is suppliedto the biomass. The biologically required amount of nutrients can bedetermined from the growth yield data, carbon load to the reactor andthe amount of each nutrient present in biomass (Wastewater Engineering:Treatment, Disposal, and Reuse. Metcalf and Eddy, Inc. 3^(rd) Edition.McGraw-Hill, Inc., New York, 1991).

[0127] Briefly, ammonia addition was gradually withdrawn, starting atabout 8 weeks prior to the scheduled shut down. The PHA levels weremonitored within the biomass on a 1-2 day basis until PHA levels reachedapproximately 20% dry weight of the biomass. The carbon influx wasstopped and the PHA levels, MLVSS, and nitrate metabolism were monitoredusing the methods as described above. The amount of PHA in the biomassdropped from 20% to about 2% in one week and a total of 121 mg ofnitrate were metabolized (see FIG. 6).

[0128] As shown in FIG. 7, PHA began to build up in the biomassapproximately 30 days after ammonia addition ceased. Withinapproximately 55-60 days (approximately 8 weeks), PHA levels rangebetween 15 and 40 weight percent. As shown in FIG. 6, approximately 20%(PHA wt/biomass wt) PHA was sufficient to support maintenance of thebiomass for 6-7 days. Therefore, prior to a scheduled shut down, it ispreferable to initiate nutrient limitation about 8 weeks in advance ofthe shut down to induce PHA accumulation.

[0129] These results demonstrate that internal PHA can function as acarbon source during periods of process waste outages, provided that thelevel of PHA in the system is followed to insure that the biomass doesnot starve. Preferably, the level of PHA in the system will be at leastthan about 1% to about 5% of the MLSS. More preferably, the level of PHAin the system will be at least than about 3% to about 5% of the MLSS.Even more preferably, the level of PHA in the system will be at leastthan about 5% of the MLSS. If the PHA level approaches about 1% to 2% ofthe MLSS, then purchased carbon sources can be supplied to keep thebioreactor viable until wastewater carbon influx is resumed.

What is claimed is:
 1. A method for monitoring and controlling thebiocatalytic efficiency of a wastewater treatment process comprising: a)providing an activated sludge environment comprising: (i) a carboninflux; (ii) cultures of autotrophic, heterotrophic and facultativemicroorganisms; (iii) a feed nutrient; and (iv) an end electron acceptorb) sampling wastewater from anaerobic, anoxic and/or aerobic stages ofthe treatment process; c) measuring the concentration of an internalstorage molecule present in the sample to determine the status of aselected sample characteristic; and d) adjusting the feed nutrient inthe activated sludge environment depending on the status of the selectedsample wherein the biocatalytic efficiency of a wastewater treatmentprocess is controlled.
 2. A method according to claim 1 wherein theinternal storage molecule is selected from the group consisting ofpolyhydroxyalkanoates and glycogen.
 3. The method according to claim 2,wherein when the concentration of polyhydroxyalkanoates is greater thanabout 15 to about 20 dry weight percent of the biomass, there is anindication that the biocatalytic efficiency of the wastewater treatmentprocess is impaired.
 4. The method according to claim 1, wherein thesampling is in situ.
 5. The method according to claim 1, wherein thesampling is continuous.
 6. The method according to claim 1, wherein thecarbon influx comprises a compound selected from the group consistingof, amines, alcohols, organic acids, carbohydrates, proteins, and aminoacids.
 7. The method according to claim 1, wherein the cultures ofautotrophic, heterotrophic and facultative microorganisms compriseorganisms selected from the group consisting of alpha, beta, and gammaProteobacteria.
 8. The method according to claim 7, wherein the alpha,beta, and gamma Proteobacteria are selected from the genera consistingof Paracoccus, Rhodococcus, Pseudomonas, Alcaligenes, Acinetobacter,Sphingamonas, Azoarcus, and Burkholderia.
 9. The method according toclaim 1, wherein the feed nutrient is selected from the group consistingof nitrate, ammonia, sulfate, sulfide, urea and phosphate.
 10. Themethod according to claim 1, wherein the end electron acceptor isselected from the group consisting of oxygen, nitrate, nitrite, nitrousoxide, ferric oxide, and sulfate.
 11. The method according to claim 2,wherein the polyhydroxyalkanoates are comprised of compounds selectedfrom the group consisting of hydroxybutyrate and hydroxyvalerate. 12.The method according to claim 1, wherein the sample characteristic isselected from the group consisting of denitrification efficiency,nitrate concentration, ammonia concentration, sulfate concentration,phosphate concentration, and carbon dioxide concentration.
 13. A methodof maintaining a viable culture in an activated sludge environment inthe absence of carbon influx comprising: a) providing an activatedsludge environment comprising: (i) a carbon influx; (ii) cultures ofautotrophic, heterotrophic and facultative microorganisms; (iii) a feednutrient; and (iv) an end electron acceptor; b) removing the feednutrient from the activated sludge environment while continuouslymonitoring the concentration of polyhydroxyalkanoates present in theactivated sludge environment; c) removing the carbon influx from theactivated sludge environment when the concentration ofpolyhydroxyalkanoates is greater than about 15 to about 20 dry weightpercent of the biomass; d) adding a minimal concentration of nitrate tothe activated sludge environment of step (c); wherein the cultures ofautotrophic, heterotrophic and facultative microorganisms are maintainedin a viable state in the absence of a carbon influx.
 14. The methodaccording to claim 13, wherein the feed nutrient is selected from thegroup consisting of nitrate, ammonia, sulfate, sulfide, urea andphosphate.
 15. The method according to claim 13, wherein the carboninflux is removed from the activated sludge environment when theconcentration of polyhydroxyalkanoates is greater than about 20 dryweight percent of the biomass.
 16. The method according to claim 13wherein the removing the feed nutrient of step (b) occurs from about 8to about 2 weeks prior to the removal of the carbon influx of step (c).